Cancers have altered transcriptional programs that lead to deregulation of normal cellular functions and support the cancer cell characteristics. A prerequisite for the cancer cell is an increase in its ribosynthetic activity to support the increased protein synthetic needs. These processes are governed by RNA polymerase I (Pol I), which is highly responsive to external stimuli and commonly hyperactivated in cancer. Yet, it has not been exploited as a clinically relevant target. Pol I transcription and ribosome biosynthesis are exquisite processes of the nucleolus. Aberration of Pol I transcription leads to nucleolar stress and disintegration of the nucleolus leading to a distinctive, identifiable cellular phenotype. This project sets forth a dual cell-based high-content imaging screen, adaptable to high-throughput screening (HTS) platforms, for regulators of nucleolar stress and of Pol I. The screen will utilize, as identifiable marks, the translocation of a nucleolar fluorescent fusion protein stably integrated in cancer cells and the expression of the catalytic subunit of Pol I. Data presented here show that the Pol I catalytic subunit can be selectively targeted for degradation and that its targeting is associated with decreased cancer cell viability. This project aims to define the feasibility of these readouts for HTS platforms, and to demonstrate the suitability and reproducibility of the assay to identify novel regulators. The rationale for the proposed work is that it will provide fundamentally novel screening tools for regulation of Pol I activity. The goal is to employ the screen to identify, firstly, inhibitors of nucleolar activities and secondly, specific regulators of the Pol I catalytic subunit. Towards these goals, in Aim 1, the dual-screening assay will be established, tested and parameters will be set for hit thresholds. Using a defined chemical library of pharmaceuticals in current therapy use, Aim 2 has been devised to systematically screen for drugs and compounds that affect the nucleolar integrity and Pol I transcription, and to provide verification of the assay set-up and reproducibility for high-throughput platforms. Aim 3 has been designed to validate the screen hits through secondary screens and assays that determine which Pol I complex factors and nucleolar processes are affected. These approaches will define the current drug space affecting Pol I, identify potential new effects beneficial for therapeutic outcome, and support application of the screen to diverse large-scale chemical library collections. The study will significantly increase the understanding how inhibition of rRNA transcription contributes to curb growth of cancer cells, and identify new drugs and small-molecule lead molecules in this process.